Throughout this application, various publications, patents, and published patent applications are referred to by an identifying citation. Corresponding complete citations are provided below under the heading "References." The disclosures of the publications, patents, and published patent specifications referenced in this application are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.
Blood is the fluid that circulates in the blood vessels of the body, that is, the fluid that is circulated through the heart, arteries, veins, and capillaries. The function of the blood and the circulation is to service the needs of other tissues: to transport oxygen and nutrients to the tissues, to transport carbon dioxide and various metabolic waste products away, to conduct hormones from one part of the body to another, and in general to maintain an appropriate environment in all tissue fluids for optimal survival and function of the cells. See, for example, Guyton, 1991.
Blood consists of a liquid component, plasma, and a solid component, cells and formed elements (e.g., erythrocytes, leukocytes, and platelets), suspended within it. Erythrocytes, or red blood cells account for about 99.9% of the cells suspended in human blood. They contain hemoglobin which is involved in the transport of oxygen and carbon dioxide. Leukocytes, or white blood cells, account for about 0.1% of the cells suspended in human blood. They play a role in the body's defense mechanism and repair mechanism, and may be classified as agranular or granular. Agranular leukocytes include monocytes and small, medium and large lymphocytes, with small lymphocytes accounting for about 20-25% of the leukocytes in human blood. T cells and B cells are important examples of lymphocytes. Three classes of granular leukocytes are known, neutrophils, eosinophils, and basophils, with basophils accounting for about 65-75% of the leukocytes in human blood. Platelets (i.e., thrombocytes) are not cells but small spindle-shaped or rodlike bodies about 3 microns in length which occur in large numbers in circulating blood. Platelets play a major role in clot formation.
Plasma is the liquid component of blood. It serves as the primary medium for the transport of materials among cellular, tissue, and organ systems and their various external environments, and it is essential for the maintenance of normal hemostasis. One of the most important functions of many of the major tissue and organ systems is to maintain specific components of plasma within acceptable physiological limits.
Plasma is the residual fluid of blood which remains after removal of suspended cells and formed elements. Serum is the fluid which is obtained after blood has been allowed to clot and the clot removed. Serum and plasma differ primarily in their content of fibrinogen and several components which are removed in the clotting process. Plasma may be effectively prevented from clotting by the addition of an anti-coagulant (e.g., sodium citrate, heparin) to permit handling or storage. Plasma constitutes about 4% of total body weight in humans. It is composed primarily of water (approximately 90%), with approximately 7% proteins, 0.9% inorganic salts, and smaller amounts of carbohydrates, lipids, and organic salts. See, for example, Carlson, 1991.
The protein portion of plasma and serum is a mixture of a large number different protein components. Standard methods, such as precipitation by various salts or organic compounds, electrophoresis, ultracentrifugation, ion-exchange chromatography, gel filtration, and immunoprecipitation with antibody-containing antisera, have been variously employed to identify and characterize at least 100 distinct protein components in human plasma. See, for example, Putnam, 1975-1987; Handin et al., 1995. Five major fractions of blood protein (as determined electrophoretically) are albumin, .alpha.1-globulin, .alpha.2-globulin, .beta.-globulin, and .gamma.-globulin.
In human blood, approximately one-half of blood protein is albumin, a relatively small protein with molecular weight of 69,000. Albumin contributes greatly to the colloid osmotic pressure of plasma and thus plays a major role in the regulation of intravascular volume and the fluid exchange between the vascular system and extravascular system. Albumin also serves as a transport protein for various substances, including small ions such as calcium and iodine and organic compounds such as bilirubin.
In human blood, the .alpha.1-globulin fraction contains proteins such as .alpha.1-acid glycoprotein, .alpha.1-antitrypsin, and .alpha.1-lipoprotein. The .alpha.2-globulin fraction contains proteins such as .alpha.2-macroglobulin, haptoglobulin, ceruloplasmin, and group-specific complement. The .beta.-globulin fraction contains proteins such as transferrin, hemopexin, .beta.1-lipoprotein, .beta.2-microglobulin, and complement components. The .gamma.-globulin fraction contains proteins such as immunoglobulins and C-reactive protein.
Immunoglobulins (which are antibodies found circulating in the blood) represent approximately one-sixth of the total human blood protein and largely constitute the .gamma.-globulin fraction. Of the different classes of immunoglobulins which can be distinguished, the principle ones are IgG, IgM, IgA, IgD, and IgE.
In addition to albumin and immunoglobulins, lipoproteins are another class of blood components and account for approximately 10% of total human blood protein. Lipoproteins are water soluble complexes comprising protein components (e.g., apolipoproteins) and lipid components (e.g., cholesterol, cholesteryl esters, phospholipids, and triglycerides). Lipoproteins are often conveniently considered to comprise a hydrophobic core (primarily of cholesteryl esters and triglycerides) surrounded by a relatively more hydrophilic shell (primarily apolipoproteins, phospholipids, and unesterified cholesterol) projecting its hydrophilic domains into the aqueous environment. Lipoproteins presumably serve as transport proteins for lipids, such as triacylglyercols, cholesterol (and cholesteryl esters), and other lipids (e.g., phospholipids). Lipoprotein remnants are the biological byproducts produced in the metabolism of lipoproteins.
Three classes of lipoproteins, .alpha.1-lipoprotein, pre-.beta.-lipoprotein, and .beta.1-lipoprotein, can be distinguished in human blood, according to their electrophoretic behavior. However, lipoproteins are more conveniently characterized by their ultracentrifligation behavior in high-salt media, as described by their flotation constants (densities), as follows: chylomicra, less than 1.006; very low density (VLDL), 1.006-1019; low density (LDL), 1.019-1.063; high density (HDL), 1.063-1.21; very high density (VHDL), &gt;1.21. Lipoproteins are often approximately spherical in shape, and range in diameter from about 0.1 micron (for chylomicra) to about 5 nanometers (for VHDL). Lipoproteins range in molecular weight from 200 kd to 10,000 kd and from 4 to 95% lipid (the higher the density the lower the lipid content). The very low- and low-density fractions appear in the .beta.1-globulin fraction and the high-density and very high-density fractions appear in the .alpha.1-globulin fraction. Chylomicra and VLDLs are rich in triglycerides (.about.90% and .about.60% of the total lipid content, respectively), while LDLs are rich in cholesterol (.about.60% of total lipid content) and HDLs are rich in phospholipids (.about.50% of total lipid content). See, for example, Converse et al., 1992.
Apolipoproteins are the protein component of lipoproteins. Examples of apolipoproteins which have been isolated from human blood and characterized include apolipoproteins A-1, A-2, A-4, B-48, B-100, C, D, and E.
Triacylglycerols (also known as triglycerides or neutral fats) are electrically neutral esters of glycerol and which possess acyl groups that are often derived from fatty acids. Examples of fatty acids include long-chain fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, palmitolic acid, oleic acid, linoleic acid, linolenic acid, and arachidoneic acid. Cholesterol triglyceride is a triglyceride where one or more acyl group is derived from cholesterol. Other glycerol esters include monoacylglycerols and diacylglycerols.
Cholesterol, the most abundant steroid in animal tissues, is usually present in blood as a lipid component of a lipoprotein. Cholesterol is a biological precursor of five major classes of steroid hormones (the progestagens, glucocorticoids, mineralocorticoids, androgens, and estrogens) which are also usually present in blood as a lipid component of a lipoprotein (i.e., they are also associated with a lipoprotein complex). Examples of such steroids include progesterone, cortisol, aldosterone, testosterone, and estrone. Cholesteryl esters, such as the apolipoproteins A1, B, C, and E, are examples of other cholesterol derivatives.
Phospholipids are lipids which comprise phosphorus; often phospholipids also contain nitrogen. Examples of phospholipids include those derived from glycerol (i.e., phosphoglycerides), such as phosphatidyl cholines, phosphatidyl ethanolamines, phosphatidyl serines, phosphatidyl inositols, and phosphatidyl glycerols. Other examples of phospholipids include those derived from sphingosine (i.e., sphingolipids), such as ceramides and sphingomyelins.
Glycolipids, another class of blood components, are lipids which comprise at least one carbohydrate group (e.g., sugar residue). Most glycolipids can be classified as glycosphingolipids (which are derived from sphingosine) or glycoglycerols (which are derived from glycerol). Mammalian glycolipids are usually derived from sphingosine. Many glycolipids may be further classified as neutral-, sulfo-, or phospho-glycosphingolipids or -glycoglycerols. Examples of simple glycosphingolipids include the cerebrosides in which there is only one sugar residue, often glucose, lactose or galactose. Examples of more complex glycosphingolipids include the gangliosides, which possess a branched chain of as many as seven sugar residues, as well as hematosides, globosides, and trihexosylceramide. See, for example, Walborg, 1978.
A number of blood proteins function as carriers for specific substances and are often referred to as transport proteins. These include apolipoproteins, discussed above, which form lipoprotein complexes with lipids, and thus are believed to facilitate the transport of these lipids. Other transport proteins include those which transport metal ions, such as the iron-binding protein, transferrin, and the copper-binding protein, ceruloplasmin, and 9.5 S-.alpha.1-glycoprotein. Prealbumin and the thyroxin-binding globulin transport the thyroid hormone, and transcortin transports the steroid hormones. Hemoglobin is eliminated from the circulation by haptoglobin, and heme is bound to hemopexin. The retinol-binding globulin binds vitamin A. The transcobalamins I, II, and III bind vitamin B12. Gc-globulin binds vitamins D2 and D3.
A number of blood proteins are enzymes, pro-enzymes, or enzyme inhibitors. Blood proteins which are enzymes (e.g., proteinases) include, for example, cholinesterase, ceruloplasmin, plasminogen, protein C, and .beta.2-glycoprotein I. Pro-enzymes (i.e., zymogens) are converted to enzymes by the action of specific enzymes. Proteinase inhibitors control this process by reducing or eliminating the activity of these specific enzymes. The major proteinase inhibitor found in human blood is .alpha.1-antitrypsin (i.e., .alpha.1-proteinase inhibitor; .alpha.1-trypsin inhibitor, prolastin) which protects tissues from digestion by elastase. Another class of proteinase inhibitors found in human blood are the antithrombins, such as antithrombin III, which prevent the effects of thrombin. Still another proteinase inhibitor found in human blood is C1-esterase inhibitor, which reduces or eliminates the activity of C1-esterase, which is the activated first component of complement, C1. Other blood proteins which are enzyme inhibitors include .alpha.1-antichymotrypsin, inter-.alpha.-trypsin inhibitor, .alpha.2-macroglobulin, and .alpha.2-antiplasmin.
A number of blood proteins are involved with the clotting process (i.e., coagulation factors). Blood clots are formed by an enzymatic cascade, with the activated form of one factor catalyzing the activation of the next factor which results in a large amplification and a rapid response to trauma. Examples of inactivated and activated clotting factors include, for example, XII and XIIa; XI and XIa; IX and IXa; X and Xa; VII and VIIa; II (prothrombin) and Ia (thrombin); I (fibrinogen) and Ia (fibrin). Other clotting factors include kininogen, kallikrein, and factors VIII, VIIIa, V, Va, XIII, and XIIIa. A number of clotting factors are also referred to as vitamin K dependent proteins, including, for example, Factor II (prothrombin), Factor VII, Factor IX, Factor X, Protein C, and Protein S.
A number of blood proteins are complement components and together comprise the complement system, which lyses microorganisms and infected cells by forming holes in their plasma membrane. More than 15 complement proteins are known, including C1, C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8 and C9.
Proteins which comprise at least one carbohydrate group (e.g., sugar residue) may be classified as glycoproteins (often also referred to as mucoproteins). The term seromucoid is often used to refer to glycoproteins derived from blood serum. In glycoproteins, typically one or more sugar residues are attached to the protein via the side chain of an amino acid residue. Many blood proteins, and virtually all plasma proteins except albumin, are glycoproteins. For example, .gamma.-globulins (e.g., immunoglobulins), .alpha.1-globulins, and .alpha.2-globulins, are glycoproteins. Examples of glycoproteins which have been isolated from human blood include .alpha.1-acid glycoprotein, .alpha.2-glycoprotein, .alpha.2-macroglobulin, .alpha.2-HS-glycoprotein, .alpha.1-antichymotrypsin, .alpha.1-antitrypsin, fibrinogen, fibronectin, pre-albumin, hemopexin, haptoglobin, transferrin, ceruloplasmin, many clotting factors, and many components of the complement system. Glycoproteins are found in substances such as orosomucoids (primarily .alpha.1-acid glycoprotein), mucin (a cell secretion), and ovomucoids (derived from egg white), which contain high quantities (e.g., about 30-50%) of carbohydrates. See, for example, Lennarz, 1980.
In addition, the liquid fraction of blood also contains a number of other components, such as electrolytes, carbohydrates, lipids, and organic salts. Electrolytes help regulate the osmotic pressure and pH of plasma. The primary cations are sodium, potassium, calcium, and magnesium. The primary anions are chloride, bicarbonate, phosphate, sulfate, and organic acids. The liquid fraction of blood also contains other small molecules (many of which are bound to proteins while transported in the blood), such as sugars (e.g., glucose), free amino acids (e.g., glutamine, alanine, glycine, and lysine), urea, uric acid, creatinine, pyruvic acid, non-esterified fatty acids (i.e., free fatty acids, FFA), bilirubin, vitamins (e.g., vitamin A), steroid hormones, and small peptides (e.g., angiotensin and bradykinin).
Human blood is a source material for the preparation of a number of blood products for clinical use. See, for example, Lawrence et al., 1996. Such blood products include, for example, whole blood (WB), red blood cells (RBC), platelets (PLT), leukocytes, fresh frozen plasma (FFP), plasma, serum, cryoprecipitated antihemophilic factor (CRYO), Factor VIII concentrates, Factor IX concentrates (prothrombin complex), albumin, plasma protein fraction (PPF), immune serum globulin (ISG), and hyperimmunoglobulins (RhD).
Human blood plasma is a valuable source material for the preparation many of these blood products. A number of organizations throughout the world are involved in the fractionation of human plasma into a variety of derivatives intended for clinical use. Among the products licensed for use in the United States are albumin, plasma protein fraction (PPF), immunoglobulin for intravenous or intramuscular injection, antihemophilic factor, Factor IX complex (the vitamin K-dependent factors), coagulation Factor IX, .alpha.1-antitrypsin (i.e., .alpha.1-proteinase inhibitor), and antithrombin III. Other products available in Europe include fibrin sealant, von Willebrand factor, Factor XIII, and C1-esterase inhibitor. See, for example, Drohan, 1994. Albumin, which is used as a plasma volume expander, is one of the major products of plasma fractionation. PPF is an albumin-rich fraction of plasma of lower purity obtained by a simpler fractionation scheme; it is more economical to produce than albumin and can be recovered in higher yield.
Other blood products include isolated immunoglobulins. Immunoglobulins are prepared from the plasma of unselected normal donors, while hyperimmunoglobulins are prepared from the plasma of donors with high antibody titers against specific antigens (e.g., tetanus, hepatitis B, Rh-D blood group antigen, and rabies). These hyperimmune donors may be identified during convalescent periods after infection or transfusion, or they may be specifically immunized to produce the desired antibodies. The immunoglobulins are usually administered intramuscularly. Intravenous immunoglobulin (IGIV) products have been developed using a variety of methods to remove or inactivate any anticomplementary aggregates. The development of IGIV has permitted the administration of much higher dosages, with a subsequent expansion in immunoglobulin therapy. See, for example, Drohan, 1994, at Chapter 138. Established indications for intravenous immunoglobulin include primary antibody deficiencies (such as congenital agammaglobulinemia, common variable immunodeficiency, X-linked agammaglobulinemia, severe combined immunodeficiency, Wiskott-Aldrich syndrome), idiopathic thrombocytopenic purpura (ITP), and B-cell chronic lymphocyte leukemia (CLL). Other conditions in which clinical benefit has been reported are secondary immunodeficiencies (multiple myeloma, protein-losing enteropathy, nephrotic syndrome), Kawasaki syndrome, treatment of viral diseases (such as human immunodeficiency virus (HIV) and cytomegalovirus infections), burn therapy, and prevention of graft-versus-host disease in bone marrow recipients. See, for example, Dwyer, 1992.
The development of coagulation factor concentrates has resulted in dramatic increases in the life expectancy and the quality of life of patients with hemophilia. Factor VIII and Factor IX concentrates are available for the treatment of hemophilia A and B, respectively. In addition, some Factor VIII concentrates contain appreciable amounts of von Willebrand factor (vWF) and may be used for the treatment of von Willebrand disease, whereas Factor IX complex concentrates can be used for the treatment of other congenital or acquired deficiencies of vitamin K dependent plasma proteins. Other plasma proteins combine to form a fibrin sealant which is used in a variety of surgical situations for its hemostatic and adhesive properties. The first component is derived from human plasma and contains fibrinogen, Factor XIII, fibronectin, and small amounts of plasminogen and other plasma proteins. The second component is a thrombin solution of sufficient concentration to clot the fibrinogen rapidly. See, for example, Drohan, 1994.
The proteinase inhibitors present in human plasma play critical roles in the regulation of the proteolytic cascades of the coagulation, fibrinolytic, complement, and kinin systems. Proteinase inhibitor concentrates have been developed to treat diseases caused by hereditary deficiencies of .alpha.1-antitrypsin (i.e., .alpha.1-proteinase inhibitor), antithrombin III, and C1-esterase inhibitor. Patients with hereditary deficiencies of .alpha.1-antitrypsin inhibitor develop pulmonary emphysema and liver disease. Clinical studies have shown that antithrombin III (AT-III) concentrates are effective in the prophylaxis or treatment of thromboembolic disorders in patients with hereditary AT-III deficiency. C1-esterase inhibitor can be used to treat hereditary angioedema, an autosomal-dominant disease that is characterized by episodic swelling of the subcutaneous tissues and the mucosa of the gastrointestinal and respiratory tracts. See, for example, Drohan, 1994.
The preparation of blood products from blood typically involves a combination of separation steps. For example, in the preparation of blood products derived from plasma, whole blood is typically first processed to removed suspended cells and formed elements (e.g., by centrifugation) to yield blood plasma. Alternatively, blood serum may be obtained by forming a blood clot (e.g., initiated by the addition of thrombin and calcium ion) and subsequently removing the clot (e.g., by centrifugation). The methods described below for the processing of blood plasma are also generally applicable to the processing of blood serum.
In the preparation of blood products and the isolation and characterization of blood components derived from blood plasma, substantial resources are expended in the field of plasma fractionation; that is, in methods for dividing plasma into the fractions rich in particular blood components, and fractions poor in particular blood components. See, for example, Curling, 1980.
Historically, plasma fractionation was achieved by salt precipitation typically using mineral salts such as ammonium sulfate and sodium sulfate; the proteins were precipitated as a result of an increase in salt concentration. In the 1940's, Cohn et al. developed an effective alternative precipitation method which employed an organic solvent, ethanol. For example, in the well known "Cohn's Method 6" (Cohn et al., 1946), ethanol is added to plasma at specific conditions of pH and temperature to obtain a specific ethanol concentration, and the resulting precipitate separated from the supernatant, and the precipitate retained. The supernatant is again treated with ethanol using different specific conditions, and the resulting second precipitate separated from the second supernatant, and the precipitate retained. The process is repeated stepwise, so that the resulting second supernatant is similarly processed, yielding a third precipitate and a third supernatant, and so on, thus yielding a sequence of precipitates (e.g., Cohn Precipitates I, II+III, IV-1, IV-4, and V; Cohn Supernatants I, II+III, IV-1, IV-4, and V), each rich in particular plasma components.
A number of other methods of fractionation by precipitation have been developed, including, for example, the use other precipitation agents such as ammonium sulfate, Rivanolg, caprylic acid, ether, and polyethylene glycol. Cryoprecipitation (e.g., the formation of a precipitate from a solution by cooling) has also been used to prepare blood fractions, most importantly a plasma cryoprecipitate rich in Factor VIII. In recent years, chromatographic methods (e.g., ion exchange chromatography, affinity chromatography, and gel filtration) have been developed to effect fractionation. In many cases, two or more fractionation methods are employed stepwise to effect the desired fractionation.
Liquid-solid separations are critical in virtually all plasma fractionation procedures in order to harvest desired components and remove contaminants. Filtration and centrifugation are two methods typically used to effect liquid-solid separations. In many instances both methods are used, stepwise, to achieve the desired separation.
Many plasma processing procedures employ filtration through relatively coarse filter media to clarify the liquid (e.g., prefiltering), often using a depth filtration process. For example, plasma may be filtered through asbestos sheet filters, often in combination with diatomaceous earth as a filter precoat. A variety of such filters are commercially available (e.g., from Seitz.RTM., Ertel.RTM., Alsop.RTM., Cellulo.RTM.). More recently, plasma has been clarified using alternative cationic depth filters comprising cellulose and diatomaceous earth treated with a cationic charge modifier. A variety of such filters are commercially available (e.g., from AMF Cuno.RTM.). Other (e.g., anionic) materials suitable for clarification are also known and commercially available (e.g., from Millipore.RTM. and Pall.RTM.).
Many plasma processing procedures also employ filtration through relatively fine filtration media. Such fine filtration media include membrane filters having pore diameters of about 0.1 to 10 microns. A variety of such filters are commercially available (e.g., from Millipore.RTM., Gelman Sciences.RTM., Nuclepore.RTM., Pall.RTM., Sartorius.RTM.&). For example, sterile filtration typically involves passage of the liquid through one or more membrane filters having pore diameters of about 0.2 microns or less, in order to exclude microorganisms, such as Pseudomonas bacteria.
Since most plasma processing procedures employ at least one precipitation or chromatographic fractionation step, these procedures invariably include further steps to eliminate the precipitating or eluting agents and to concentrate the final product. Methods for achieving one or both of these goals include lyophilization, thin film evaporation, gel permeation, and ultrafiltration. Thin film evaporation methods are typically used to remove organic solvents. Gel permeation methods are typically used to remove precipitating agents, such as ethanol and salts, but yield the desired product in a dilute solution.
Ultrafiltration (i.e., nanofiltration, dialysis) is often performed to achieve one or both goals. In many cases, gel permeation in combination with ultrafiltration is employed to obtain the desired product purity and concentration. Ultrafiltration methods employ ultrafine filtration media, including, for example, flat membranes, spiral membranes, hollow fiber systems, and tubular systems. A variety of such filter materials are commercially available (e.g., from A/G Technology.RTM., Millipore.RTM.). Common ultrafiltration membranes are comprised of hollow fiber filters or flat sheet filters (e.g., comprising polyvinylidene fluoride, polysulfone, and/or cellulose ester) having an effective size exclusion of about 25-500 nanometers, or about 1,000 to 500,000 kd.
Filtration (e.g., of plasma, plasma fractions, or partially purified blood products) through fine and/or ultrafine filtration media is invariably hampered by rapid clogging (e.g., fouling) of the filtration media resulting in a low, often impractical, flow rate through the media, as well as increased costs. For example, if plasma protein fraction (i.e., PPF) is filtered directly through a 0.2 micron or tighter membrane filter, the throughput is very low and a large amount of the membrane is required to filter even relatively small amounts of PPF. Blood components (e.g., lipids, lipid complexes, lipid-like materials, colloids) often cause filtration and separation problems, such as fouling, clogging or otherwise impairing (e.g., "blinding off") filtration and separation media, such as ultrafiltration media and/or chromatographic media. However, it has been found that the materials which cause clogging can often be removed by a pre-filtration step through relatively coarse filtration media (as described above), thereby permitting a high throughput through fine and/or ultrafine filtration media, and thus increasing efficiency and reducing costs.
Although pre-filtration methods have increased the efficiencies of fine filtration and ultrafiltration, other methods have been examined. For example, pre-treatment methods for the removal of materials (e.g., lipids, lipid complexes, lipid-like materials, colloids) which cause clogging of the filtration media have been investigated.
For example, a common pre-treatment method involves the use of colloidal fumed silica at a rate of about 40 grams per liter of liquid being treated (i.e., .about.0.04 g/mL) (Condie, 1979). Lipoproteins, cholesterol, and triglycerides are adsorbed on the colloidal fumed silica, and the resulting residue is removed by centrifugation. Unfortunately, a large proportion of the liquid (20-50% of the starting volume) remains trapped within the centrifuged precipitate. This trapped volume often contains significant quantities of the product sought, and is difficult to recover. Additionally, the colloidal fumed silica is not very selective, and significant amounts of albumin and serum immunoglobulins are removed along with the lipids.
Another pre-treatment method involves the use of tricalcium phosphate (i.e., Ca.sub.3 (PO.sub.4).sub.2) at a rate of about 20-50 grams per liter of liquid being treated (i.e., .about.0.02-0.05 g/mL) (Burstein et al., 1957). It is believed that the interaction between many lipids (e.g., chylomicra and .beta.1-lipoproteins) and calcium results in precipitation. The resulting colloidal slurry is then centrifuged to remove the tricalcium phosphate and bound lipids.
Yet another pre-treatment method involves the precipitation of lipoproteins from plasma by the addition of dextran sulfate (i.e., a heparin-like polysaccharide containing up to three sulfate groups per sugar residue) at a rate of about 0.1-0.5% w/v (i.e., 0.001-0.005 g/mL). The resulting lipid-rich precipitate is then removed by centrifugation. However, the clarified serum or plasma then contains dextran sulfate, and additional processing is required for its removal.
The present invention provides methods for the selective separation of organic components (e.g., lipids, lipid complexes, lipid-like materials, colloids, and components thereof) from biological fluids containing them, which methods comprise the step of contacting the biological fluid with a synthetic hydrated alkaline earth silicate (i.e., SHAES), such as synthetic hydrated calcium silicate (i.e., SHCS) or synthetic hydrated magnesium silicate (i.e., SHMS). In one embodiment, the methods of the present invention are useful as pre-treatment methods in the preparation of blood products and in the isolation and characterization of blood components derived from blood.
Synthetic hydrated alkali earth silicates have been described in methods for the processing of certain liquids. For example, hydrated calcium silicate has been described in methods for the removal of colloids and color forming materials in the purification of sugar solutions (Bottoms, 1951). Hydrated calcium silicates have also been described as a sweetener for organic dry cleaning solvents to remove free fatty acids (i.e., FFA) which accumulate upon repeated usage (Riede, 1963). Compositions comprising diatomaceous earth, synthetic calcium silicate hydrate, and synthetic magnesium silicate hydrate have been described in methods for the reduction of the free fatty acid (i.e., FFA) content and color degradation in cooking oils used in fast food outlets and other restaurants (see, for example, Duensing et al., 1978). Synthetic hydrated calcium silicates have been described as drying agents in methods for drying delactosed or deproteinized solutions obtained as by-products in the concentration of whey proteins and casein (Lauck et al., 1983). Base-treated magnesium silicates have been described as adsorbents in methods for the removal of contaminants such as free fatty acids (i.e., FFA), soaps, phosphorus, metal ions, and color bodies from glyceride oil (Denton, 1993). Synthetic hydrated alkaline earth silicates have been described as absorbents (e.g., for converting liquids or low melting point solids to free flowing powders) and as fillers, carriers, flatting agents, decolorizers, and catalyst carriers (Celite, 1991).
The present disclosure represents the first use of synthetic hydrated alkaline earth silicate (i.e., SHAES) in methods for the preparation, isolation, and/or characterization of biological products for clinical use, and in particular, for organic components of mammalian blood.